Many diseases are characterized by the development of progressive immunosuppression in the patient. The presence of an impaired immune response in patients with malignancies has been particularly well documented. Cancer patients and tumor-bearing mice have been shown to have a variety of altered immune functions such as a decrease in delayed type hypersensitivity, a decrease in lytic function and decreased proliferative response. S. Broder et. al., N. Engl. J. Med., 299, 1281 (1978), E. M. Hersh et. al., N. Eng. J. Med., 283, 1006 (1965).
Many other diseases are also characterized by the development of an impaired immune response. Progressive immunosuppression has been observed in patients with acquired immunodeficiency syndrome (AIDS), sepsis, leprosy, cytomegalovirus infections, malaria, etc. The mechanisms responsible for the down-regulation of the immune response, however, remain to be elucidated.
T lymphocytes (T cells) are critical in the development of all cell-mediated immune reactions. Helper T cells control and modulate the development of immune responses. Cytotoxic T cells (killer T-cells) are effector cells which play an important role in immune reactions against intracellular parasites and viruses by means of lysing infected target cells. Cytotoxic T cells have also been implicated in protecting the body from developing cancers through an immune surveillance mechanism. T suppressor cells block the induction and/or activity of T helper cells. T cells do not generally recognize free antigen but recognize it on the surface of other cells. These other cells may be specialized antigen-presenting cells capable of stimulating T cell division or may be virally-infected cells within the body that become a target for cytotoxic T cells.
Cytotoxic or suppressor T cells usually recognize antigen in association with class I Major Histocompatibility Complex (MHC) products which are expressed on all nucleated cells. Helper T cells, and most T cells which proliferate in response to antigen in vitro, recognize antigen in association with class II MHC products. Class II products are expressed mostly on antigen-presenting cells and on some lymphocytes. T cells can be also divided into two major subpopulations on the basis of their cell membrane glycoproteins as defined with monoclonal antibodies. The CD4.sup.+ subset which expresses a 62 kD glycoprotein usually recognize antigen in the context of class II antigens, whereas the CD8.sup.+ subset expresses a 76 kD glycoprotein and is restricted to recognizing antigen in the context of Class I MHC.
The CD4.sup.+ subset can be further subdivided into two functionally distinct groups. One group of cells positively influences the immune response of T cells and B cells. The second group of cells induces suppressor/cytotoxic functions in CD8.sup.+ cells.
The definitive T-cell marker is the T-cell antigen receptor (TCR). TCR-2 is a heterodimer of two disulfide-linked polypeptides (.alpha. and .beta.) while TCR-1 is structurally similar but consists of .gamma. and .delta. polypeptides. Both TCR-1 and TCR-2 are associated with a complex of polypeptides which comprise the CD3 complex.
The TCR found on the surface of all T cells is composed of six or seven different subunits which can be divided into three distinct subgroups of proteins. R. D. Klausner et. al., Annu. Rev. Cell Biol., 6, 403 (1990). One subgroup of proteins comprises the .zeta. family dimers. Three proteins, encoded by two genes, appear to comprise the .zeta. mily. These proteins are .zeta., its alternately spliced form, .eta., and the .gamma. chain of multisubunit Fc.gamma. receptors. The heterodimers .alpha..beta. or .gamma..delta. within the receptor complex are responsible for ligand binding. The heterodimer is found on most mature T cells and the .alpha..beta. heterodimer is found predominantly on T cells that are .gamma..delta. located in epithelia.
The final subgroup of proteins which comprise the TCR are the CD3 chains which encompass three distinct, but closely related subunits. These subunits are the glycoproteins .gamma. and .delta. and the non-glycosylated protein .epsilon.. The CD3 chains are encoded by three homologous, clustered genes. F. Koning et. al., Eur. J. Immunology, 20, 299 (1990); R. S. Blumberg et. al., Proc. Natl. Acad. Sci. USA, 87, 7220 (1990). Diversification of receptor types is the result of segregation of chains of the TCR complex into multiple subunits. Incompletely assembled complexes are degraded, resulting in the surface expression of only completely assembled receptors. R. D. Klausner, New Biol., 1, 3 (1989).
T-cell recognition events lead to signal transduction and appropriate biochemical signals that control cellular responses. The ability of TCR to transduce signals to multiple biochemical cascades is the central event of immune cell activation. The details of this signal transduction pathway, however, are poorly understood. For the TCR, one or more tyrosine (Tyr) kinases likely have an essential role in T-cell activation. R. D. Klausner et. al., Cell. 64. 875 (1991). At least two signal transduction pathways are activated upon stimulation of TCR by antigen or by monoclonal antibodies directed against CD3 or the .alpha..beta. heterodimer. Stimulation of TCR activates a tyrosine kinase. L. E. Samelson et. al., Cell 46, 1083 (1986); M. D. Patel et. al. J. Biol. 262, 5831 (1987); E. D. Hsi et. al., J. Biol. Chem. 264, 10836 (1989). Phosphorylation of several proteins on tyrosine residues is induced within seconds of TCR stimulation. C. H. June et. al., J. Immunol. 144, 1591 (1990). None of the TCR chains possesses intrinsic kinase activity. The tyrosine kinase Fyn, however, coprecipitates with the CD3 complex. L. E. Samelson et. al., Proc Natl. Acad. Sci. USA. 87, 4358 (1990). The T-cell-specific member of the Src family of tyrosine kinases, Lck, is tightly, but noncovalently, associated with the cytoplasmic domain of either the CD4 or CD8 molecule. The extracellular domains of CD4 and CD8 bind to MHC class II and class I molecules, respectively. Upon binding of TCR to an antigen-MHC complex on a presenting cell, the TCR would be brought into close proximity with either a CD4 or CD8 molecule that could independently bind to the appropriate MHC molecule.
TCR also activates a phosphatidylinositol-specific phospholipase C which leads to hydrolysis of phosphatidylinositol-4,5,-bis-phosphate. A. Weiss et. al., Proc. Natl. Acad. Sci. USA. 81, 4169 (1984); J. B. Imboden et. al., J. Exp. Med. 161, 446 (1985). This leads to the liberation of two second messengers. Inositol-1,4,5-tris-phosphate is responsible for transient Ca.sup.+2 mobilization. Diacylglycerol is a potent activator of protein kinase C. B. Berridge et. al., Nature, 341, 197 (1989).
The cytoplasmic domain of the TCR .zeta. chain is sufficient to couple stimulation of the receptor with the signal transduction pathways. B. A. Irving et. al., Cell. 64, 891 (1991). A chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the .zeta. chain was constructed. The chimeric protein activated the appropriate signal transduction pathways in the absence of CD3 .gamma., .delta., and .epsilon.. Therefore the role of CD3 .zeta. is to couple the TCR to intracellular signal transduction mechanisms.
The identification and isolation of soluble mediators of the immune response has heightened interest in the development of clinical trials using immunotherapy as a form of treatment. Interleukin-2 (IL-2), a lymphokine produced by helper T cells, stimulates the growth of T cells that bear IL-2 receptors, either in vivo or in vitro. The in vitro incubation of resting lymphocytes in supernatants containing IL-2 for three to four days results in the generation of lymphocytes capable of mediating the lysis of fresh tumor cells, but not normal cells. These cells are referred to as lymphokine activated killer (LAK) cells. I. Yron et. al., J. Immunol. 125. 238 (1980); M. T. Lotze et. al., Cancer Res. 41, 4420 (1981); and S. A. Rosenberg et. al., J. Natl. Cancer Inst., 75, 595 (1985).
The activation of T lymphocytes to generate T-activated killer cells (T-AK) has been described as taking lymphocytes by leukophoresis or from peripheral blood, and stimulating said cells with a monoclonal antibody (MAb) to a T cell surface receptor such as anti-CD3 (soluble or solid phase bound). The T cells can be stimulated with or without addition of one or more cytokines such as IL-2. Alternatively, T cells can be purified before stimulation with the MAb to a surface receptor. Experimentation with T-AK cells has demonstrated that CD8.sup.+ cells are responsible for the non-MHC restricted cytolytic activity seen in these cultures. P. M. Anderson et. al., J. Immunol. 142. 1383 (1989); C. M. Loeffler et. al., Cancer Res, 51, 2127 (1991). The ability of IL-2 to expand T lymphocytes having immune reactivity and the ability to lyse fresh autologous, syngeneic, or allogeneic natural killer (NK) cell-resistant tumor cells, but not normal cells, has resulted in the development of cell transfer therapies.
Typical adoptive immunotherapy involves the administration of immunologically active cells to an individual for the purpose of providing a beneficial immunological effect such as reduction or control of cancer. These immunologically active cells are typically taken by venipuncture or leukophoreses either from the individual to be treated, as in autologous treatment, or from another individual, as in allogeneic treatment. The lymphocytes are then cultured to increase their number and to activate their antitumor activity, and then infused back into the patient. Thus, the majority of conventional efforts in adoptive immunotherapy are directed at expanding cells in vitro followed by infusion back into the patient.
Animal experiments involving the transfer of immunologically active cells from healthy animals to animals with cancerous tumors have indicated that adoptive immunotherapy can elicit an antitumor effect in certain tumor models with a high degree of effectiveness. The administration of IL-2 together with LAK cells has proven effective in the treatment of a variety of murine malignancies. The transferred LAK cells also proliferate in vivo as a result of IL-2 treatment. Human clinical trials have demonstrated that LAK cells plus IL-2 or IL-2 alone can be effective in mediating the regression of established metastatic cancer in selected patients. S. A. Rosenberg, "Immunotherapy of Patients with Advanced Cancer Using Interleukin-2 Alone or in Combination With Lymphokine Activated Killer Cells" in Important Advances in Oncology 1988, J. B. Lippincott Co., 217, (1988).
The success of adoptive immunotherapy has been limited by the large number of cells required in the therapy, the large amount of culture medium and large number of hours involved in culturing cells to develop LAK activity, the length of time sufficient LAK activity must be maintained for the desired therapeutic efficacy, the time involved in clinical treatment and the side effects of treatment. Improvements in the in vitro culturing process have been made in order to increase the efficacy of adoptive immunotherapy. Cells cultured in IL-2 and/or monoclonal antibodies against the antigen receptor complex CD3 (anti-CD3 MAb) have been found to induce proliferation of a greater number of T cells, which demonstrate an increased anti-tumor activity. P. M. Anderson et. al., Cancer Immunol. Immunother. 27, 82 (1988); P. M. Anderson et. al., J. Immunol. 142, 1383 (1989); and A. C. Ochoa et. al., Cancer Res. 49, 963 (1989).
There has been limited success with efforts to activate in vivo antitumor mechanisms. Only a minority of patients receiving high doses of IL-2 experienced therapeutic effects and significant toxicity is observed. The direct infusion of anti-CD3 monoclonal antibody alone induces nonspecific antitumor function in mice. D. W. Hoskin et. al., Cancer Immunol Immunother., 29, 226 (1989). Based on the positive results in murine models, direct infusion of anti CD3 has been attempted in humans. Although patients who have directly received the anti-CD3 MAb OKT3 have experienced the activation of some T cells in vivo. The toxicity of intravenous OKT3 reaches the maximum tolerated dose (MTD) at low doses before it starts showing some immune efficacy. W. Urba et. al., Cancer Res., in press. It is believed that the free OKT3 is responsible for the majority of these toxic effects.
T lymphocytes from hosts bearing tumors exhibit decreased immune function in a variety of in vitro tests. R. Lafreniere et. al., J. Surg. Oncol. 43, 8 (1990); R. J. North et. al., J. Exc. Med. 159, 1295 (1984); M. Sarzotti et. al., Int. J. Cancer. 39, 118 (1987). It has been observed that before the decrease in the immune responsiveness in peripheral blood lymphocytes, T lymphocytes infiltrating tumor exhibit poor cytotoxic activity against autologous or allogeneic tumor cells. E. F. Klein et. al., 1980, In: Contemporary Topics in Immunobiology, I. P. Witz and M. G. Hanna, Jr., eds. Plenum Press, N.Y., p 79-107; B. M. Vose et. al., J. Cancer, 44, 846 (1981).
The molecular basis of the decreased immune responsiveness of the T cells is poorly understood. It has been proposed that decreased immune responsiveness of the T cells is caused by the development of suppressor lymphocytes. S. B. Mizel et. al., Proc. Natl. Acad. Sci. USA. 77, 2205 (1980); C. C. Ting et. al., Int. J. Cancer. 4, 644 (1979). Another proposal is that responsive T-cell clones are deleted. S. Webb et. al., Cell. 63, 1249 (1990). It has also been proposed that decreased immune responsiveness of the T-cells is the result of the induction of T-cell energy. M. K. Jenkins et. al., J. Exp. Med. 165, 302 (1987). Others have suggested that the major alteration in the immune response is produced by a modification in the presentation of the antigen which
results in an inadequate response of the CD4.sup.+ helper T lymphocytes. These data have been strengthened by the observation that tumor cells transfected with cytokine genes induce a protective antitumor response, and result in an immunological memory response. E. R. Fearon et. al., Cell. 60: 397 (1990).
Augmentation of the immune response in immune compromised patients via infusions of lymphokines and/or adoptive immunotherapy has met with variable success. In view of the fact that the immune system of the patient being treated may already be suppressed, a need exists for effective methods of measuring the progression of immunosuppression so that attempts at augmenting the immune system can be effectively timed. A need exists for a method by which the patient's level of immuno-suppression can be estimated and on the basis of which the patient's likely response to therapy can be predicted accurately and the patient's therapeutic plan can be developed. A method is needed by which the clinician can determine whether the patient's T lymphocytes will be capable of activation and, thus, whether autologous adoptive immunotherapy will likely be efficacious. A need exists for a method by which the immunosuppressed state of T lymphocytes during disease progression can be circumvented or reversed so that the T cell immune response in the patient can develop or be augmented. A need also continues to exist for a method of screening for immunosuppressive agents and agents that reverse or inhibit immunosuppression.